0270.6474/84/0404-1118$02.00/O The Journal of Neuroscience Copyright 0 Society for Neuroscience Vol. 4, No. 4, pp. 1118-1129 Printed in U.S.A. April 1984 CORTICOTROPIN-RELEASING FACTOR: CO-EXPRESSION WITHIN DISTINCT SUBSETS OF OXYTOCIN-, VASOPRESSIN-, AND NEUROTENSIN-IMMUNOREACTIVE NEURONS IN THE HYPOTHALAMUS OF THE MALE RAT’ P. E. SAWCHENK0,2 L. W. SWANSON, AND W. W. VALE The Salk Institute for Biological Studies and The Clayton Foundation for Research-California Division, La Jolla, California 92037 Received October 11,1983; Revised November 28,1983; Accepted November 28, 1983 Abstract Two immunohistochemical methods that allow the concurrent localization of neuroactive sub- stances within individual neurons have been used to identify, count, and chart the distribution of corticotropin-releasing factor (CRF)-immunoreactive cells in the paraventricular nucleus of the hypothalamus (PVH) that may also contain an additional peptide. In colchicine-treated male rats a moderate number of oxytocin-stained cells, localized primarily in a discrete, anterior part of the magnocellular division of the nucleus, was found also to stain positively for CRF. Similarly, oxytocin and CRF immunoreactivity were jointly expressed in magnocellular neurons distributed diffusely in the supraoptic nucleus. Smaller numbers of vasopressin- and neurotensin-stained neurons centered in specific parts of the parvocellular division of the PVH were stained with antisera against CRF. Possible mechanisms whereby the function of subsets of magnocellular and parvocellular neurose- cretory neurons can be modulated differentially are discussed. The recent isolation and characterization (Vale et al., hormone to portal vessels in the median eminence are 1981) of a 41-residue peptide that appears to correspond localized in the paraventricular nucleus of the hypothal- to the long-elusive corticotropin-releasing factor (CRF) amus (PVH). This nucleus is best known for its role in has had obvious physiological and clinical implications. the synthesis and delivery of the nonapeptide hormones Antisera to the peptide have also provided morphologists oxytocin and vasopressin to the posterior lobe of the with a marker for the group of parvocellular neurosecre- pituitary, although it is now clear that the PVH also tory neurons that stimulates the release of ACTH and contains separate and topographically distinct subpopu- P-endorphin from the anterior lobe of the pituitary gland. lations of neurons that project to the neurohemal zone Immunohistochemical studies on the distribution of of the median eminence and to autonomic centers in the CRF-stained neurons (e.g., Bloom et al., 1982; Bugnon brainstem and spinal cord (see Swanson and Sawchenko, et al., 1982; Olschowka et al., 1982; Cummings et al., 1983). In a recent analysis of the distribution of CRF- 1983; Merchenthaler et al., 1983; Swanson et al., 1983) immunoreactive neurons in the rat brain (Swanson et confirm recent functional work (e.g., Makara et al., 1981; al., 1983), it was noted that stained cells in the PVH are Ixart et al., 1982; Antoni et al., 1983) suggesting that localized primarily in discrete parts of the parvocellular while the peptide is widely distributed in the central division of the nucleus, where their distribution overlaps nervous system, the vast majority of cells delivering the in part with that of several other neuropeptides, includ- ing neurotensin (Kahn et al., 1980). In addition, a smaller i This work was supported by Grants NS-16686, DA-0029, and AM- number of CRF-stained cells were found in parts of the 26741 from the United States Public Health Service and was conducted magnocellular division of the PVH in which oxytocin- in part by the Clayton Foundation for Research-California Division; containing cells are known to be concentrated (Rhodes P. E. S., L. W. S., and W. W. V. are Clayton Foundation Investigators. et al., 1981a; Sawchenko and Swanson, 1982). In view of We wish to thank Ms. Donna Chin for preparing histological material, the apparent overlap in the distribution of the neuropep- Ms. Pat Thomas for typing the manuscript, and Mr. Kris Trulock for photographic help. We are especially grateful to Dr. M. Brown for tides, as well as a large body of evidence indicating that providing an antiserum against neurotensin, Dr. J. Rivier for synthetic oxytocin and (especially) vasopressin may serve to mod- peptides used in blocking experiments, and Dr. F. Vandesande and Dr. ulate ACTH secretion (see Yates and Maran, 1974; Vale K. Dierickx for antisera against oxytocin and vasopressin. et al., 1983), we have examined the possibility that CRF ’ To whom correspondence should be addressed. is co-expressed along with other peptides in individual 1118 The Journal of Neuroscience Co-localization of CRF Immunoreactivity 1119 neurons of the PVH. Other recent analyses have failed the PVH, in which staining for both antigens was ade- to provide a consistent view of this problem; CRF-im- quate and controls for effective elution were negative, munoreactive neurons in the magnocellular division of were generated for the oxytocin-CRF comparison (n = the PVH have been reported to contain either oxytocin 4), the vasopressin-CRF comparison (n = 3), and the (Burlet et al., 1983) or vasopressin (Roth et al., 1983a) neurotensin-CRF comparison (n = 3). Over 50 partial or neither peptide (Bugnon et al., 1982; Antoni et al., series, generated from 23 rats, provided confirmation of 1983). the basic results described below. As an additional control for method specificity, eight Materials and Methods animals were pretreated and perfused as described above, and small blocks of hypothalamic tissue were cut serially Tissue preparation and immunohistochemical proce- in sections 5 to 6 pm thick. The members of pairs of dure. Adult male albino rats of the Sprague-Dawley individual sections at all levels of the PVH were stained strain were used in all experiments. Most animals re- individually for the presence of oxytocin and CRF, or ceived a single injection of 50 to 100 pg of colchicine into vasopressin and CRF, or neurotensin and CRF. Compar- the lateral ventricle 48 to 72 hr before perfusion to arrest isons of the location and morphology of stained neurons axonal transport and thereby enhance the immunohis- in adjoining pairs of sections were again made photo- tochemical staining of neuropeptides in cell bodies. This graphically. pretreatment is necessary to demonstrate maximal num- Antisera and controls. Antisera against oxytocin and bers of CRF- and neurotensin-immunoreactive neurons vasopressin were provided by Dr. F. Vandesande and Dr. in the PVH. The animals were perfused with ice-cold 4% K. Dierickx (University of Ghent, Belgium) and were paraformaldehyde in a two-phase procedure in which the cross-adsorbed in the solid phase against the heterolo- pH of the perfusate is varied (Sawchenko et al., 1982; gous antigen according to a procedure outlined elsewhere Swanson et al., 1983). Up to five one-in-five series of 20 (Swaab and Pool, 1975). Specific staining with each pm thick frozen sections through the PVH were saved antiserum was blocked when immunoprocessing was car- and then prepared for indirect immunofluorescence ried out using sera that had been pre-incubated with an staining of cells that cross-react with antisera against excess (15 mg ml-l) of the homologous synthetic peptide CRF, neurotensin, oxytocin, or vasopressin using a con- and then diluted to the working concentration of 1:2000. ventional method (Sawchenko and Swanson, 1981) based The antiserum against neurotensin was provided by upon the localization of a primary antiserum raised in Dr. M. R. Brown (Peptide Biology Laboratory, The Salk rabbits using an affinity-purified, fluorescein-conjugated Institute) and was prepared against neurotensin conju- goat anti-rabbit IgG. The distributions of labeled cells gated with glutaraldehyde to thyroglobulin. This serum were mapped onto projection drawings made from adja- (Nil-1) is directed against the C-terminal portion of the cent series of sections counterstained with thionin, and molecule (Brown et al., 1978) and was used at a dilution the region around the PVH was photographed at x 100 of 1:4000. All staining in the hypothalamus was blocked magnification using Ilford XP-1 film. The reaction prod- by the addition of synthetic neurotensin (15 mg ml-l) to uct was then eluted from the tissue with a variant of the the serum. method described by Tramu et al. (1978). In brief, the To ensure the validity of our localization of CRF- sections were rinsed in phosphate-buffered saline (PBS) immunoreactive neurons, three sera against CRF human and then placed in a freshly prepared solution of 0.15 M a-globulin conjugates were used. Two of these (C24 and KMn04 and 0.01 N H2S04 for 30 to 60 sec. They were C30) are directed against the C- and N-terminal portions, then transferred immediately to 0.5 M NaHS03 until respectively, of the ovine CRF molecule (Swanson et al., decolorized and were rinsed thoroughly (3 x 10 min) in 1983; Vale et al., 1983). The third was prepared against PBS. The sections were then reincubated in fluorescein- a synthetic rat CRF (Rivier et al., 1983) conjugate. Each labeled secondary antiserum, rinsed (2 X 10 min) in PBS, of these sera was used at a final dilution of 1:3000, and and inspected. The presence of any immunoreactivity in pre-incubation of each with 22 mg ml-l of the respective these sections was taken as evidence that the elution immunogen blocked specific staining of cells and fibers procedure had been less than maximally effective, and in the hypothalamus. All sera used for both normal the material was either re-eluted or discarded. Sections immunohistochemistry and blocking experiments con- in which the elution procedure was judged to have been tained 10 mg ml-’ of the “carrier” protein used for complete were then processed for the demonstration of immunization. a second neuropeptide using the protocol described Finally, although there has been no suggestion from above, except that the incubations were carried out on our previous radioimmunoassay (Vale et al., 1983) or slide-mounted sections.